A. Field of the Invention
The invention relates to a method and apparatus for computer-implemented processing and manipulation of an image in order to extract data from the image, and more specifically, the present invention relates to a method and apparatus for manipulation of a master pattern and a scanned image in order to identify information present in the scanned image by comparing the scanned image to a modified master pattern.
B. Description of Related Art
In the field of electrophoretic separations of macromolecules and two-dimensional electrophoretic separations, proteins and other biomolecular matter are separated for identification and quantitative analysis. Such two-dimensional procedures typically involve sequential separations by iso-electric focusing (IEF) and sodium dodecyl sulfate (SDS) slab gel electrophoresis using gel media for both iso-electric focusing and SDS electrophoresis.
A protein is a macromolecule composed of a chain of amino acids. Some amino acids found in proteins carry a negative charge and others carry a positive charge, in some pH range. A specific protein, defined by its specific sequence of amino acids, is thus likely to incorporate a number of charged groups along its length. The magnitude of the charge contributed by each amino acid is governed by the prevailing pH of the surrounding solution, and can vary from a minimum of 0 to a maximum of 1 charge (positive or negative depending on the amino acid), according to a titration curve relating charge and pH according to the pK of the amino acid in question. Under denaturing conditions in which all of the amino acids are exposed, the total charge of the protein molecule is given approximately by the sum of the charges of its component amino acids, all at the prevailing solution pH.
Two proteins having different ratios of charged, or titrating, amino acids can be separated by virtue of their different net charges at some pH. Under the influence of an applied electric field, a more highly charged protein will move faster than a less highly charged protein of similar size and shape. If the proteins are made to move from a sample zone through a non-convecting medium (typically a gel such as polyacrylamide), an electrophoretic separation will result.
If, in the course of migrating under an applied electric field, a protein enters a region whose pH has that value at which the protein""s net charge is zero (the isoelectric pH), it will cease to migrate relative to the medium. Further, if the migration occurs through a monotonic pH gradient, the protein will xe2x80x9cfocusxe2x80x9d at this isoelectric pH value. If it moves toward more acidic pH values, the protein will become more positively charged, and a properly-oriented electric field will propel the protein back towards the isoelectric point. Likewise, if the protein moves towards more basic pH values, it will become more negatively charged, and the same field will push it back toward the isoelectric point. This separation process, called iso-electric focusing, can resolve two proteins differing by less than a single charged amino acid among hundreds in the respective sequences.
Typically, iso-electric focusing is performed using a tube or column of medium with a prepared tissue sample deposited at the top of the tube. Current is applied between opposite ends of the tube causing the proteins in the tissue to migrate to their respective isoelectric focusing points. The medium in the tube now includes proteins that have been separated linearly along the length of the medium in the tube. The medium and proteins are next removed from the tube and loaded along one edge of an SDS slab gel for further electrophoresis. The SDS slab gel is typically a rectangular assembly that includes a separation medium retained between to glass plates. Current is applied between opposite edges of the SDS slab gel thereby causing the proteins from the tube to further separate along the planar orientation of the slab gel. The glass plates are then removed from the medium and separated proteins and stained. The staining process makes the proteins visible to the naked eye revealing a two dimensional pattern of spots.
An automated apparatus and method for 2-D separations of proteins is described in greater detail in U.S. Pat. No. 5,993,627 to Anderson et al. and is incorporated by reference in its entirety. U.S. Pat. No. 5,993,627 includes a detailed description of sample preparation, electrophoresis procedures and staining of 2-D gels such that a plurality of separated proteins are visible after staining. Specifically, after staining, each separated protein produces a dark spot that is visible to the naked eye and can further be photographed by a camera or other video medium such as a computer imaging scanner. FIG. 1 is an electronically scanned image of one such 2-D gel showing a plurality of spots, each spot representing a protein or in some areas, a pair of proteins that migrated to the same isoelectric focusing point.
Serious study has been given to the relative locations of separated proteins on 2-D gels. Specifically, it is well known that from any give tissue sample, the proteins of that sample will migrate during the electrophoretic process to specific locations on the 2-D gel relative to other proteins thereby defining a recognizable pattern of spots. For instance, a sample taken from tissue of a rat""s liver subjected to a reproducible 2-D electrophoretic process produces a recognizable pattern of spots, each spot formed by one or possibly two proteins. Repeated study of gel spot patterns for various tissues has subsequently led to the development of master patterns of spots, at least one master pattern for each tissue, where each master pattern visually represents the specific relative locations of the spots (relative to each other) within the recognizable pattern of spots. An example of a master pattern is shown in FIG. 2.
Although not shown in FIG. 2, information for each spots includes: a unique identifying number (also known as a master spot number or MSN), an (x,y) coordinate identifying the relative center of the identified spot on an (x,y) grid assigned to the gel image; a x-axis width, a y-axis width, and computer pixel intensity of the center of the spot. In addition to the recognizable pattern of spots, identification of many of the individual proteins at numerous spot locations has been added to each master pattern. Specifically, many of the spots in the gel pattern have been analyzed to determine the exact molecular composition of that protein.
A master pattern may be based upon the study of a healthy tissue, a treated tissue (treated with pharmaceuticals), diseased tissue and/or combinations thereof. In other words, the master pattern may include spot specific information obtained from the combined results of study of healthy tissue, treated tissue and diseased tissue or portions thereof.
The study of new pharmaceutical products often requires comparison of treated animal tissue with untreated animal tissue. Using the master pattern for guidance, a comparison can then be made of 2-D gels produced from treated tissues with other 2-D gels from a comparison set. The 2-D gels produced from treated tissues can yield important data concerning the effects of the new pharmaceutical on the tissues being studied. Specifically, some proteins in the treated tissues may not be present in the master pattern (untreated tissue) and conversely, some proteins in the master pattern (untreated tissue) may not be present in the treated tissue. Additionally, differences in the amount of protein between treated and control samples can yield important information about the effects of the pharmaceutical on the tissue.
Similarly, the study of diseased tissue also often requires comparison of the diseased tissue with healthy tissue. 2-D electrophoresis gels produced from healthy tissue are typically used to generate a master pattern for that tissue. A comparison can then be made between 2-D gels produced from diseased tissue and 2-D gels from healthy tissue. Some proteins in the healthy tissues may not be present in the diseased tissue and conversely, some proteins in the diseased tissue may not be present in the healthy tissue, and there may be quantitative differences in amount of certain proteins. Such information provides markers for detecting the corresponding disease.
One tool used for analyzing a 2-D gel involves image manipulation by a computer to warp two images into alignment with one another. Warping refers to a process of applying geometric corrections to modify the shape of features in an image and to change their spatial relationships. Another term used for a warping process is rubber-sheeting because the warping process can be likened to stretching a rubber sheet wherein portions of one or more images are stretched or shrunk in order to bring the spots on all the images into registration with one another and still maintain relative positional relationships between the spots.
Although images have been warped together to provide visual information about a 2-D gel, there is a need for technology wherein a master pattern is warped to an image in order to provide information about a 2-D gel based upon the information in the master pattern.
One object of the invention is to provide a reliable method and apparatus for providing spot specific data concerning the spots in a 2-D electrophoresis gel.
In accordance with one aspect of the present invention, a method for processing visual information from an image of a 2-D electrophoresis gel includes having a master pattern in digital form, scanning the image thereby converting the scanned image from visual information into digital form, comparing the scanned image with information from the master pattern, and outputting numeric data based upon the comparison.
Preferably, the visual information includes a plurality of spots defining a pattern of spots, each spot representing at least one protein.
Preferably, an object pattern is created from the scanned image and spot data in the master pattern is matched with spots represented in the object pattern. Using the matched spots, the master pattern is warped into alignment with the object pattern thereby creating a new object pattern from the master pattern. The new object pattern is augmented with spots in the object pattern not present in the master pattern. The parameters of the spots in the new object pattern are then modified by an optimization procedure to fit a processed image derived from the scanned image.
Preferably, the outputted information includes spot specific data such as:
an indication of presence of spots in the scanned image not found in the master pattern;
an indication of the absence of spots in the scanned image found in the master pattern;
an indication of the master spot number for spots in the new object pattern;
an indication of the location of spots in the new object pattern;
an indication of x and y dimensions of spots in the new object pattern;
an indication of the intensity of spots in new object pattern; and
an indication of the integrated density of spots in the new object pattern.
In another aspect of the present invention, a computer is programmed to perform the steps of the method described above.